The present invention relates to methods for separating particles or molecules by migration in a separation medium.
A large number of methods are already known in which separation is obtained by applying a motive force within a separation medium to particles or molecules to be separated.
If the motive force is of the electrical type, the situation is referred to as electrophoresis, and if it is of hydrodynamic origin, the situation is referred to as chromatography or filtration.
Chromatography, filtration and electrophoresis are very widely used for purifying or analyzing molecules or macromolecules, whether synthetic or natural, particles, cells, organelles or viruses. There are large number of ways of implementing these methods, descriptions of which may be found in various works, for example xe2x80x9cChromatographie en phase liquide et supercritiquexe2x80x9d, R. Rosset, M. Caude, A. Jardy, Masson ed., Paris 1991; xe2x80x9cPractical High Performance Liquid Chromatography, V. R. Meyer, John Wiley ed. Chichester, N.Y. USA; xe2x80x9cChromatography of polymersxe2x80x9d, T. Provder ed., ACS publ. Washington D.C., 1993 or xe2x80x9cElectrophoresis: theory, techniques and biochemical and clinical applications, A. T. Andrews, Oxford University Press, N.Y. 1986xe2x80x9d.
However, these techniques have limitations. For DNA, for example, chromatography only allows separation for molecules of relatively small size, and agarose or acrylamide gel electrophoresis is most often preferred to it.
Constant-field electrophoresis does not itself make it possible to separate molecules larger than a few tens of thousands of base pairs (kilobases, Kb), while the intact chromosomes of most cellular organisms, prokaryotic or eukaryotic, and much viral DNA, are several hundreds of kilobases or several megabases (millions of base pairs, Mb) long. Human DNA, for example, has sizes between 50 and 200 Mb. For a large number of applications in medicine and genetics, such as genetic (genome) mapping, variability analysis, cloning with artificial yeast chromosomes, diagnosis, etc., DNA exceeding the separation limits of constant-field electrophoresis needs to be separated as a function of its size.
In order to solve this problem, a new technique known as pulsed field gel electrophoresis has been proposed (PCT WO 84/02001, inventors C. Cantor and D. C. Schwartz, 24.05.84). A large number of variants of this technique has also been developed (see for example EP 0 356 187, EP 0 256 737, U.S. Pat. No. 4,971,671, EP 0 395 315, xe2x80x9cPulsed filed [sic] Electrophoresisxe2x80x9d, B. Birren, E. Lai, Academic Press, London 1993; xe2x80x9cPulsed filed [sic] Gel electrophoresisxe2x80x9d, Meth. in Mol. Biol., M. Burmeister and L. Ulanovsky Eds, Humana Press, Totowa, N.J. USA, 1992, and the references cited in these patents and works).
In spite of its significant success, this technique, too, still has drawbacks. The main one is its slowness: it takes several days to separate chromosomes containing several megabases. In addition, since the separation is carried out in a gel, it is difficult to recover the DNA after separation, and this method is poorly suited to preparative applications. Lastly, it remains limited to sizes smaller than 10 Mb.
Electrophoresis can also be used to separate particles of micron or submicron size (colloidal particles, cells, viruses, red blood cells or white blood cells, etc.), as may be necessary in sample analysis, in purification, for diagnosis or for treating certain diseases.
If the particles to be separated have different surface potentials, they can be separated in a liquid medium as a function of this surface potential. Conversely, for a large number of applications it is desirable to separate them as a function of the size of the particles or molecules having the same surface potential. This cannot be done in a liquid medium, and it has been proposed to carry out this type of separation by electrophoresis in very dilute agarose gel, but the particles have a tendency to become trapped in the gel, and these gels are very difficult to handle. Further, this method only works for relatively small particles, typically smaller than one micrometer (G. A. Griess, P. Serwer, Biopolymers, 29, 1863-1866 (1990)). In this case as well, pulsed field electrophoresis makes it possible to extend the range accessible to the method slightly, but in a limited way. Lastly, it should be pointed out that it does not make it possible for particles of similar size to be separated satisfactorily.
In summary, although methods are already known for separating particles, large molecules and in particular DNA, these methods have a large number of drawbacks, linked in particular with their slowness and the difficulties which are encountered with these methods for carrying out the separation of molecules or particles of large size.
One object of the invention is to provide a separation method which does not have these various drawbacks.
Separation methods are already known which employ a combination of electric and magnetic fields in order to separate particles, and in particular DNA.
These methods, referred to as electromagnetophoresis, have been described for example in U.S. Pat. No. 4,726,904 and also in the following publications:
Mukherjee, H. G., majumdar, D., xe2x80x9cFresenius""Zxe2x80x9d Anal. Chem., 277, 205 (1975),
O. Lumpkin, J. Chem. Phys. 92, 3848-3852 (1990)
Kowalczuk, J. S., Acta Chromatogr. 1, 34-55 (1992).
In these methods, the magnetic field supplements the electric field to cause migration of the charges to be separated.
U.S. Pat. No. 4,526,681 has already proposed a technique for separating magnetic particles, according to which the particles to be separated are introduced into a ferrofluid medium, to which a magnetic fields [sic] is applied which makes it possible to distribute said particles along a magnetic susceptibility gradient.
However, this technique can only be applied for the separation of magnetic particles having different magnetic susceptibilities.
For its part, the invention provides a method for separating particles or molecules, in which these particles or molecules are introduced into a separation medium which is a ferrofluid, that is say a colloidal suspension of magnetic particles, and at least one motive force is applied to these particles or molecules within said ferrofluid, characterized in that a magnetic field is applied to this ferrofluid which creates in it at least one alternation of one (or more) zone(s) rich in and a zone lean in magnetic particles that the particles or molecules pass through during their migration, which brings about their separation.
A method of this type makes it possible to separate the particles or molecules as a function of the speed at which they move within the ferrofluid, and performs better than the methods known to date in terms of separation speed and/or in terms of the size range in which the separation can be carried out.
It will be noted that the method proposed by the invention makes it possible to separate essentially nonmagnetic particles (minimal or zero magnetic susceptibility).
The motive or migration force used is generally nonmagnetic.
In a preferred embodiment of the invention, which is particularly advantageous for the separation of particles carrying an electric charge, the migration force is obtained by applying an electric field within the separation medium. The method then constitutes an electrophoresis method.
It will in general be advantageous to choose as the separation medium a ferrofluid whose magnetic particles are essentially neutral, so that they will not be moved under the action of the electric field. However, certain particular applications may nevertheless require magnetic particles of given charge, if it is desired for example to decrease or increase the interaction of the particles to be separated with the particles.
In a preferred embodiment, which may or may not be combined with the preferred embodiments described above, the magnetic field is essentially perpendicular to the direction of motion of the particles.
Two embodiment versions which are moreover preferred, and may be combined with any one of the embodiments described above but are mutually exclusive, consist in using a magnetic field:
a) which is essentially constant in the zone where it is applied,
b) which has an intensity gradient in the zone where it is applied.
Similarly, two embodiment versions which may be combined with any one of the embodiments described above but are mutually exclusive, consist in using:
c) a separation zone which has a thickness which is essentially constant in the direction of the magnetic field, this thickness being chosen, with the concentration of the magnetic particles in the separation fluid and the amplitude of the magnetic field, as a function of the dimensions of the particles or molecules to be separated; in particular, larger thicknesses are preferably used for particles and molecules of larger size;
d) the separation zone has a variable thickness along the preferential migration direction of the particles or molecules to be separated, the thickness of this zone being chosen, with the concentration of the magnetic particles in the separation fluid and the amplitude of the magnetic field, as a function of the dimensions of the particles and molecules to be separated; in particular, larger thicknesses are preferably used for particles and molecules of larger size.
The methods according to paragraphs a) and c) are preferably used to obtain a high resolution over a relatively small range of sizes, while the methods according to paragraphs b) and d) are better suited to separations in a large range of sizes.
In a particularly simple embodiment variant of paragraph d), the walls of the separation zone which are essentially perpendicular to the magnetic field are slightly inclined relative to one another, giving said zone a xe2x80x9cwedgexe2x80x9d shape.
In the favored embodiments described above, the best results are most often obtained when the average dimension of the separation zone in the direction parallel to the direction of the magnetic field is between 1 micrometer and 1 mm, and preferably between 10 micrometers, and 100 micrometers.
This method is advantageously implemented with the various following steps:
filling the separation zone with the separation medium;
activating the magnetic field;
introducing a certain quantity of a sample containing the particles or molecules to be separated on one side of the separation zone;
activating means exerting a motive force on the particles or molecules to be separated.
It should be noted that the order in which these steps are listed corresponds to a favored embodiment of the invention, but that it is equally possible in the scope of the invention to implement them in a different order, for example by activating the magnetic field after the introduction of the samples to be separated, and/or by activating rather the motive force.
In the scope of the invention, detection or observation of the passing of the separated products and/or collection of the separated products may be implemented at the outlet of the separation zone.
Advantageously, in addition, the ferrofluid is automatically replaced between two separation operations.
The invention is particularly advantageous for the separation of particles or macromolecules of large size, such as nucleic acids and particularly DNA, and yet more particularly DNA molecules of size between 50 Kb and several hundreds of Mb. It is also particularly advantageous for the separation of objects in suspension in a liquid, such as cells, viruses, nonmagnetic colloidal suspensions and liposomes. These examples should not however be interpreted as a restriction of the field of the invention, which may also in certain cases prove advantageous for the separation of other types of objects like, here again given by way of nonrestrictive examples, proteins and synthetic or natural macromolecules.
A further object of the invention is to provide a device for the separation of particles or molecules which comprises a cell that receives a separation medium, which is a ferrofluid, that is to say a colloidal suspension of magnetic particles, means for introducing these particles or molecules into said separation medium and means for applying a magnetic field to this fluid, characterized in that it includes means for applying at least one migration force within said medium and in that, for the application of a magnetic field, there are means capable of creating at least one alternation of a zone rich in and a zone lean in ferrofluid magnetic particles that the particles or molecules to be separated pass through during their migration, which brings about their separation.
The method and the device according to the invention are advantageously used in the scope of a diagnostic method, for separating particles and molecules and, especially but not exclusively, DNA, cells, blood cells or viruses.
They may also be used for obtaining medicines, veterinary or phytosanitary products and cosmetic products, for example including liposomes, proteins, DNA, cells, blood cells, viruses or colloidal suspensions in their composition.
Other characteristics and advantages of the invention will become more apparent from the following description. This description is purely illustrative and does not imply any limitation.